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Urease Metallocenter Assembly
[edit]Overview
[edit]Urease metallocenter assembly refers to the process by which the transition metal containing active site of urease is assembled.[1] This process includes but is not limited to the incorporation of 2 Ni2+ ions into urease, and the carbamylation of the active site lysine to render the enzyme functionally active.[1] The pathway has been found to be GTP-dependent and facilitated via a cascade of accessory proteins which bind the apo enzyme and shuttle nickel to the resultant complex for insertion.[2]
Unfacilitated Metallocenter Assembly
[edit]The necessity for facilitated loading of urease with Ni2+ comes from evidence that the nickel binding site is heavily buried in the protein, and not solvent exposed.[3] Specifically, the metal cannot be removed from the enzyme using the chelating agent EDTA, unless conditions are employed to denature or unfold the protein.[3] Moreover, urease with no nickel bound does not easily take up nickel from solution, with or without denaturing conditions.[2] This information led to suggestions that nickel was entering urease via protein flexibility or additional factors in biological systems[3]
Urease Accessory Proteins
[edit]| Accessory Protein | Role | Genetic Loci Relative to Structural Genes |
|---|---|---|
| UreD (UreH in Helicobacter pylori)[2] | Scaffold Protein[2] | Upstream[3] |
| UreF[2] | Potential Fidelity Enhancer[2] | Downstream[3] |
| UreG[2] | GTPase[2] | Downstream[3] |
| UreE (not reported in eukaryotes)[2] | Metallochaperone[2] | Downstream[3] |
In the bacteria Klebsiella aerogenes and Helicobacter pylori, urease's (UreABC apoprotein) metallocenter assembly assisted by: UreD, UreE, UreF, UreG accessory proteins.[2] However, UreD is referred to as UreH in H. pylori.[2] In eukaryotes that contain urease, an UreE homologue is not known.[2] Also, despite the fact that eukaryotic urease is composed of homo-oligomers containing their own active sites and that bacterial urease is composed of multimers with 2 to 3 subunits,[4] the metallocenter sites are the same for all ureases.[2] These accessory protein's genes are typically downstream & upstream of urease's subunits in bacteria, with order and letter assignments changing depending on organism.[2] The genes for UreD, UreF, and UreG homologues are not adjacent to urease's subunit genes in eukaryotes.[2]
Metallocenter Pathway Working Models
[edit]In models of assembly for Klebsiella aerogenes, UreD, UreF, and UreG bind either to UreABC sequentially (one UreD, UreF, UreG for each of the 3 UreABC oligomers), or as the preformed heterotrimeric UreD:UreF:UreG complex.[2] Formation of the ultimate UreABC:UreD:UreF:UreG:UreE complex is facilitated by further binding of nickel-bound UreE.[2] The final complex's role includes GTP hydrolysis by UreG, Ni2+ insertion, and active site lysine carbamylation, with the accessory proteins dissociating from urease at the finish.[2] Currently, the mechanism for nickel insertion and lysine carbamylation by bicarbonate is still a subject for study.
UreD & UreH: Scaffold Proteins
[edit]Role
[edit]It is hypothesized that UreH/D's role is to act as a scaffold that binds urease to recruit the other accessory proteins, and facilitate nickel insertion into the active site.[2] The exact location of UreD binding to urease is not known.[2] Modelling attempts using experimental data have shown UreD/H binding to the UreB & UreC subunits of apo urease, with one UreD/H bound to each UreABC oligomer.[2]
Evidence to Support Role
[edit]The beta-helical fold in UreH contains 17 beta strands and 2 alpha helices.[2] This fold resembles another scaffold complex involved in metallocenter synthesis.[2] Additionally, a study with metal-free urease incubated with nickel and UreD led to 15% more functional urease than without UreD. Due to this information, it was hypothesized that UreH/D is involved in serving as a scaffold and or recruiting the other accessory proteins, and is involved in facilitating nickel insertion into the active site.[2]
Role
[edit]In Klebsiella aerogenes, UreF's role is to bind UreABC near UreB to provide a binding site for UreG and to regulate UreG's ability to catalyze GTP hydrolysis.[2] UreF exercises its regulatory role via binding interactions with the subsequently bound UreG on the opposite side of UreG's GTP site.[2] In this way, GTP hydrolysis can be efficiently coupled to urease activation.[2] UreD & UreF are both thought to bind close to UreB, the apoenzyme component of urease that could allow access to the forming active site via a hinge-like motion.[2]
Evidence to Support Role
[edit]UreF inH. pylori (PDB: 3CXN) and can form alpha-helical bridging dimers that link 2 UreHs and serve as a binding domain for UreG.[2] In this complex, a helix at the N-terminus of UreF is bound by UreH and allows for UreF's Tyr48 to become unburied.[2] The UreG binding site is the strongly conserved UreF dimer face which contains its C-terminus and Tyr48.[2]
Functionally, UreABC:UreD:UreF has similar urease activity to when UreF is not bound (UreABC:UreD).[2] Since UreF was found to bind UreG on a face opposite to UreG's GTP site in a UreH:UreF:UreG complex, it is not believed to be a GTPase-activating protein because it does not permit it to enhance activity.[2] Additionally, UreG shows enhanced GTPase activity in the complex when UreF is mutated. This information supports the idea that UreF controls GTPase activity in a way to ensure that GTP hydrolysis and metallocenter assembly are efficiently paired.[2]
UreG: GTPase
[edit]Role
[edit]The role of UreG in Klebsiella aerogenes is to bind UreABC:UreD:UreF or UreF to increase its GTPase activity such that it can hydrolyze GTP and participate in nickel insertion.[2] UreG's Asp80 has been identified as key in this binding interaction.[2] Note that UreG cannot bind the complex when UreB is absent, and that monomeric UreG contains a metal binding site where Zn2+ and Ni2+ have been found.[2] Currently, a crystal structure of free monomeric UreG is not available, likely attributed to UreG being an intrinsically disordered protein.[2]
Evidence for Role
[edit]UreG binding to apo urease (UreABC:UreD:UreF) cannot occur when it lacks UreB. Monomeric UreG also exhibits poor to no GTPase activity, as compared to when bound to the urease activation complex (UreABC:UreD:UreF:UreG).[2] Through studying the mutagenesis of residues' effects on complex formation, Asp80 was identified as key on the UreG binding region of UreF.[2] Additionally, when incubated with nickel and bicarbonate (for lysine carbamylation), 60% of present active sites in UreABC:UreD:UreF:UreG have nickel insertion and become active.[2]
Monomeric UreG of ‘’K. aerogenes’’ can bind either Ni2+ or Zn2+ in a 1:1 ratio.[2] However, the UreABC:UreD:UreF:UreG complex can form without Ni2+ in K. aerogenes, and in vitro by combining UreABC:UreD:UreF and UreG.[2]
UreE: Nickel Metallochaperone
[edit]Role
[edit]UreE in K. aerogenes is a metallochaperone that is thought to bind cytoplasmic Ni2+ and shuttle the ions to the urease activation complex for insertion via binding interactions with UreG.[2] The residues involved UreE-UreG interactions are not yet identified.[2] UreE contains a C-terminal Polyhistidine-tag region that can bind 6 Ni2+ ions.[2] Though, UreE mutants lacking this region can still contribute to urease activation.[2] When dimerized, UreE:UreE contains an interfacial metal binding site containing His96 from each UreE, and 2 peripheral metal binding sites that included His110 & His112 from each subunit.[2] The interfacial site appears to be the only metal binding site necessary for UreE function.[2]
Evidence for Role
[edit]UreABC:UreD:UreF:UreG:UreE has been found to generate completely activated urease.[2]
A UreE mutant lacking the Polyhistidine-tag (H144*UreE (PDB: 1GMW)) bound with copper, possessed 3 sites for metal binding: (1) an interfacial site containing His-96 from each UreE subunit, and (2) peripheral metal binding sites containing His110 & His112 on each UreE.[2] Mutations in the interfacial metal binding site of UreE led to inactive UreE, as compared to mutations in other sites which did not impair function.[2]
Mechanism for Metal Insertion
[edit]Currently, the exact mechanism of nickel insertion to urease from UreE via the activation complex UreABC:UreD:UreF:UreG:UreE is not established.
References
[edit]- ^ a b written; Klein, translated by Wolfgang Kaim, Brigitte Schwederski, Axel (2013). Bioinorganic chemistry : inorganic elements in the chemistry of life : an introduction and guide (Second edition. ed.). Chichester, UK: Wiley-Blackwell. ISBN 9780470975244.
{{cite book}}: CS1 maint: multiple names: authors list (link) - ^ a b c d e f g h i j k l m n o p q r s t u v w x y z aa ab ac ad ae af ag ah ai aj ak al am an ao ap aq ar as at au av aw ax ay az ba bb Farrugia, M. A.; Macomber, L.; Hausinger, R. P. (28 March 2013). "Biosynthesis of the Urease Metallocenter". Journal of Biological Chemistry. 288 (19): 13178–13185. doi:10.1074/jbc.R112.446526.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ a b c d e f g Hausinger, edited by Robert P.; Eichhorn,, Gunther L.; Marzilli, Luigi G. (1995). Mechanisms of metallocenter assembly. New York: VCH. ISBN 1-56081-920-0.
{{cite book}}:|first1=has generic name (help)CS1 maint: extra punctuation (link) - ^ Permyakov, Eugene A. (2009). Metalloproteomics. Hoboken, N.J.: John Wiley & Sons. ISBN 978-0-470-39248-5.